An unusual amino acid, hypusine, which occurs in only one cellular protein, eukaryotic translation isolation factor 5A (eIF-5A), is intimately involved in cell proliferation. Hypusine biosynthesis occurs by way of two sequential post-translational modification reactions: i) deoxyhypusine synthesis by deoxyhypusine synthase and ii) deoxyhypusine hydroxylation by deoxyhypusine hydroxylase. We have purified the first enzyme, deoxyhypusine synthase, from rat testis. The purified enzyme displays a remarkably narrow specificity toward its substrates, spermidine, NAD, and the eIF-5A precursor protein and catalyzes deoxyhypusine synthesis in the complete reaction mixture. In the absence of the substrate protein, however, it carries out a partial reaction, the NAD-dependent cleavage of spermidine. The enzyme exists as a tetramer of 42 kDa subunits, with a pI of 4.75. Using partial amino acid sequences from the rat testis enzyme, we identified YHRO68W of Saccharomyces cerevisiae chromosome VIII as the gene for deoxyhypusine synthase. Inactivation of the deoxyhypusine synthase gene causes loss of viability in yeast, providing proof that the hypusine modification is essential for eukaryotic cell proliferation. We have also cloned human cDNAs encoding a full- length deoxyhypusine synthase by immunoscreening of a HeLa cell cDNA library. After overexpression of the human deoxyhypusine synthase cDNA or the yeast cDNA of YHRO68W in E. coli, we purified the recombinant enzymes, and are characterizing their physical and enzymatic properties. Collaborations are underway to determine the three-dimensional structures of human and yeast deoxyhypusine synthase. Deletion, insertion and site-directed mutagenesis studies are being carried out to gain insights into the active site structure and the structure-function relationship of this important enzyme.